ABSTRACT Enteric infections caused by bacterial pathogens are often debilitating and life-threatening, especially for young children. In vivo rodent models and in vitro intestinal epithelial cell monolayers, the most common models for studying these pathogens, often do not recapitulate the true outcomes of enteric infections that occur in the human intestine. Therefore, many aspects of the interactions between these pathogens and the human host remain unknown. This project aims to dissect the early events between cells in the human small-intestine and two important intestinal enteric pathogens, Vibrio cholerae and Yersinia pseudotuberculosis, using a multicellular 3D in vitro human tissue model that incorporates flow, low 02 tension, immune cells and normal microbiota. Specifically, we will use silk proteins as scaffolds to develop a 3D multicellular matrix system to support human intestinal epithelium formation for sustained cultivation and for infection by enteric pathogens. These silk scaffolds will be seeded with cells from enteroids derived from children. Our preliminary data shows that these 3D silk scaffolds that are seeded with cells from ileal enteroids develop at least 4 major intestinal cell types when placed in differential medium. Building on these results, this project will continue to develop and study these intestinal tissues by sequentially introducing flow dynamics, physiological concentrations of 02 (5%), immune cells, and normal microbiota into these 3D ileal tissues scaffolds. Our goals are to understand (1) how, where and the consequences of V. cholerae and Y. pseudotuberculosis binding to and damaging the human intestine under conditions of flow and low 02; (2) the bacterial and host transcriptomes after infection of this human tissue model; (3) the impact of neutrophils on colonization, damage and host responses to infection; (4) the impact of specific Y. pseudotuberculosis adhesins and Yops on this human intestinal model; and (5) the impact of V. cholerae quorum sensing on its colonization; and (6) the impact of interspecies communication between V. cholerae or Y. pseudotuberculosis and normal microbiota on colonization and tissue damage. In addition, we will probe the utility of this model for assessing anti-infectives by testing a number of small molecules that disrupt quorum sensing or type 3 secretion systems on the colonization and damage caused by these pathogens. Establishing this tunable 3-D intestinal system will permit the development of methods that interrupt seeding, spread and/or damage by the pathogen by targeting specific bacterial mechanisms or by supplementing the host with specific microbiota or other factors that compete for the same niches, inhibit colonization, and/or reduce intestinal damage and inflammation.